KR100293017B1 - Group B Streptococcus type II and V polysaccharide-protein coupled vaccines - Google Patents

Abstract

Antigenic conjugate (A) comprising (1) a capsular polysaccharide (CPS) of type II or V G.B Streptococcus and (2) a protein (I) linked to 2 or more side chains, terminating in sialic acid residues, of CPS through a sec. amino bond is new. CPS has mol.wt. at least 5000. Pref. 5-50% of terminal sialic acid residues are available for linkage to (I); esp. about 7% or 25% for type II or type V CPS respectively. Partic. (I) are tetanus or diphtheria toxoid, or cross reactive material 197.

Description

[Name of invention]

Group B Streptococcus type II and V polysaccharide-protein coupled vaccines

[Declaration of Invention]

The present invention was made with government support under NIH grants A123339, A130628 and A128040 granted by the National Institutes of Health. The government has certain rights in this invention.

[Field of Invention]

The present invention relates to antigenic binding molecules consisting of antimolecular polysaccharides covalently bound to proteins of group B Streptococcus type II and V type. The invention also relates to vaccines and immunization methods for immunizing mammals including the human body against infection by group B Streptococcus type II (GBS II) and / or type V (GBS V). Also included are multivalent vaccines consisting of antigens for other pathogenic bacteria and binding molecules of the invention.

Background of the Invention

Infection with group B streptococcus (GBS) is the single most common cause of neonatal sepsis and meningitis in developed countries (3, 31). Recent reports from the United States have found that mortality rates (9-13%) are lower than those of the 1970s due to the recent diagnosis and enhanced treatment of Ami (1, 10). Nevertheless, fatal infections are still occurring, and still many, less than 50% of GBS meningitis survivors have chronic neurological damage across deaf and alleviated mathematics disorders to severe motor, sensory and cognitive impairment (3). Preventing rather than improved diagnosis or treatment will have the most important effect in further reducing GBS-related morbidity and mortality.

Since GBS ectopic polysaccharide-specific antibodies have been shown to protect both experimental animals (23, 24) and human infants (4, 5) from GBS infection, several polysaccharides have been purified and tested in healthy adults as experimental vaccines ( 6). If a safe and efficacious GBS vaccine is available, it may be injected into a woman before or during pregnancy to induce antibodies that are transmitted to the fetus in the uterus to protect against neonatal infection. Of these GBS polysaccharides (types Ia, II and III) tested in volunteers, type II has the highest immunogenicity and induces type II-specific antibody responses in 88% of preimmunized recipients (6). While various GBS serotypes are already known to significantly affect the proportion of patients with GBS infection, it is not known that V affects a significant proportion of GBS infections. In newborns, the level of specific antibodies required to protect against type II GBS infection is not exactly limited, but is approximately 2-3 μg / ml (6). In vaccine recipients that achieve antibody responses that are slightly above the minimum required for prevention, the amount of maternal antibodies transferred through the placenta has a half-life of maternal immunoglobulin G (IgG) in neonates, so maternal IgG is incompletely transferred to the fetus. It may be inadequate to protect premature babies. Increasing the magnification of specific antibody responses in vaccination in these two groups of patients can prevent many infants. Metastasis of maternal IgG to the fetus will increase over the third three months so that higher maternal antibody levels will be protected earlier, ie, earlier in premature infants (16). Likewise, higher levels of maternals may protect against post-onset disease by maintaining maternal antibodies in infants longer.

The immunogenicity of various bacterial polysaccharide antigens has been increased by attaching a suitable carrier protein to the polysaccharide or derivative oligosaccharide (2, 14, 17-19, 22, 27, 29, 30, 34). Preferred properties of polysaccharide-protein binding vaccines include increasing immunogenicity against polysaccharides, elevating hapten-specific antibody responses to progenitor doses, and superior IgG class antibodies. Recently, it has been successful to develop GBS III-Tetus toxin (TT) by using a side chain sialic acid group as a directional protein binding site (33). Unbound III-type polysaccharides are non-immunogenic in rabbits, whereas III-TT vaccines induce GBSIII-specific, opsonin-activated antibodies (33).

[Summary of invention]

The present invention is composed of the antimolecular polysaccharides and protein components derived from group B streptococcus type II or the group B streptococcus type V and protein components, wherein the sialic acid residues of the II and V polysaccharide components are terminal or The above-mentioned side chains are related to antigenic binding molecules, characterized in that they are respectively linked to a protein through a second amine bond.

The present invention comprises the steps of: (a) oxidizing a periodate sufficient to introduce a group B streptococcus type II or V anaerobic polysaccharide to two or more terminal sialic groups bound to the polysaccharide backbone;

(b) oxidizing the group B streptococcus type II or V anaerobic polysaccharide to form a secondary amine bond between the anaerobic polysaccharide and the protein through reductive amination to bind to the protein. B streptococcus type II or V relates to a method for producing a binding molecule of the polysaccharide.

Binding molecules prepared according to the above methods of the present invention are also included in the present invention.

The invention also relates to a vaccine consisting of each of said binding molecules. The invention also relates to a multivalent vaccine consisting of the binding molecule of the invention and at least one immunogenic molecule capable of producing antibodies against pathogenic agents other than group B Streptococcus type II or V. In particular, in addition to consisting of GBS type II and / or type V binding molecules, the multivalent vaccines according to the invention further comprise group B streptococcus Types Ia, Ib, III, IV and Haemophilus influenzae b. Or other immunogenic molecules capable of causing the production of antibodies to a hospital selected from the group consisting of type V and this coli K1 type.

In another embodiment of the present invention, the neonatal immunization method comprises a fetus pregnant before and after administration of a vaccine consisting of a binding molecule of the present invention to a woman in an amount sufficient to prevent infection when the newborn is born by administering an immunogenic amount to the female. Allows the production of antibodies that can be transferred.

The present invention also relates to a method of immunizing an adult by administering an immunogen to an adult with a vaccine consisting of the binding molecule of the present invention. The present invention also relates to a method of immunizing an adult by administering an immunogen dose to a human body that is susceptible to infection by a group B streptococcus type II or V vaccine comprising the binding molecule of the present invention.

In another embodiment of the invention, a vaccine consisting of a binding molecule of the invention administers an immunogen to a volunteer vaccine receptor that awards serum or plasma which can be administered manually to the group. The present invention also provides a method for immunizing a human body, including newborns, children and adults who are at risk of being infected by group B Streptococcus type II or V. Being at risk can include a human body with a reduced immune system for a variety of reasons, including but not limited to cancer or diabetes.

Another embodiment of the invention relates to a method for immunizing livestock against bovine mastitis consisting of administering an immunogen to a dairy cow in a vaccine consisting of the binding protein of the invention.

[Brief Description of Drawings]

1 is a structure of a heptasaccharide repeating unit of type II GBS distillate polysaccharide.

2 shows GBS type II polysaccharide competitive ELISA. GBS Type Ia (○), Type II (●), and Type III (▲) polysaccharides were used as inhibitors of II-TT vaccine antibodies that bind to plates coated with type II polysaccharides. The results are expressed as inhibition rates for control wells without inhibitors.

3 shows GBS type II polysaccharide competitive ELISA. Native (•) and desialylated or nuclear (□) II polysaccharides were used as inhibitors of II-TT vaccine antibodies. Points in the data represent the average of three measurements (with standard deviation). Results are shown as inhibition rate for control wells without inhibitor.

4 is a repeating unit structure of V-type GBS distillate polysaccharides.

5 shows GBS V-polysaccharide competitive ELISA. GBS type Ia (-▲-), type Ib (-△-), type II (-●-), type III (-○-), type V (-■-) and type VI (-□-) polysaccharides It was used as an inhibitor of the antibody caused by the V-TT vaccine. The data points are the average of two measurements. The results are expressed as inhibition rates for control wells without inhibitors.

FIG. 6 shows in vitro opsonin cell death by human peripheral blood lymphocytes of GBS V strain CJB III treated with opsonized rabbit rabbit antiserum generated against GBS V-TT binding vaccine. C 'is complement.

[Description of Preferred Embodiment]

The present invention relates to antigenic binding molecules consisting of anti-leak polysaccharide components and protein components.

Bacterial strains. GBS type II strain 18RS21 and type Ia strain 090 were obtained from the high Rebecca Lancefield of Rockefeller University and stored in freeze culture at -80 ° C. Strain 18RS21 has been used in in vitro and in vivo assays and is a raw material of type II antimicrobial polysaccharides used in binding vaccines. Two GBS type II clinical isolates (strains S16 and S20) and type III strain M781 were obtained from the Channing Laboratory's culture collection.

Previously it was not recognized that it had a significant effect on the number of GBS infections in humans, but recent evidence shows that type V serotypes contribute about 15% of GBS infections. To prepare GBS-V bound vaccines, GBS V strain 1169-NT1 was obtained from J. Jelinkova of The Institute of Hygiene And Epidomology, Prague, Czech Republic and stored in freeze culture at -80 ° C. It was. Strain 1169-NT1 was used in in vitro and in vivo assays and is the raw material of the type V anticapillary polysaccharide used in the binding vaccine. GBS type V strain CJB 111 was obtained from Dr. Baker Baker of Baylor University.

Binding of GBS II polysaccharides or V polysaccharides with TT. Type II maknae polysaccharide is purified from strain 18RS21 by the above method for the purification of III polysaccharide (33). The binding of the type II polysaccharide to the monomer TT is carried out using the technique described above to bind the TT to the GBS III polysaccharide (33). In short, natural Type II polysaccharides are fractionated in size on Sepharose CL-6B column (1.6 by 85 cm; Pharmacia Fine Chemicals). The material flowing out in the middle of the main peak is collected, dialyzed against water and lyophilized to give a material having a relative molecular weight of 200,000. This material was analyzed at 500 MHz with ¹ H-nuclear magnetic resonance spectroscopy to confirm that it was a natural type II polysaccharide structure (20) and no group B antigen (26). Polysaccharides suitable for use as the binding molecule of the invention span a very wide range of molecular weights. The preferred molecular weight range for the polysaccharide component is between about 5,000 and 1,000,000 daltons. A more preferred range is between 100,000 and 300,000. Preference is given to polysaccharides having a molecular weight of about 200,000 Daltons in the range of 100,000 to 300,000.

Size-fractionated type II polysaccharides are moderately oxidized with sodium meta- periodate (18). In this process, the sialic acid portion of the polysaccharide is changed to an 8 carbon sialic homologue, 5-acetamido 3,5-dideoxy-D-galactosyloxultoxolic acid (33). The ratio of oxidized sialic acid groups is calculated by gas chromatography-mass spectroscopy of sialic acid groups and their oxidized derivatives as described above (33). Preferably, about 5-50% of the sialic acid group of each GBS type II polysaccharide can be used to modify and bind to the protein. Most preferably about 5-25% of the sialic acid group of the GBS type II polysaccharide can be used to modify and bind to the protein. Most preferred is a modification of about 5-10% of the sialic acid group of the GBS II polysaccharide.

Oxidation of GBS V-type polysaccharides preferably modifies about 5 to 80% of the terminal sialic acid groups to form reactive aldehydes. More preferred is a modification of about 10-50% of the terminal sialic acid group.

The protein component of the binding molecule of the present invention may be any physiologically acceptable protein. Preferred proteins include tetanus toxin, diphtheria toxin and cross-reactive substances such as CRM ₁₉₇ .

The oxidized type II polysaccharide is bound to monomeric tetanus toxoid ("TT") (Institute Armand Frappier, Montreal, Canada) by reductive amination as described above (33). TT is purified into monomers by gel filtration chromatography as described above (33). In brief, 10 mg of the periodate-treated Type II polysaccharide and 10 mg of purified TT are dissolved in 0.6 ml of 0.1 M sodium carbonate (pF 8.1). Sodium cyanoborohydride (20 mg) is added to the mixture and incubated at 37 ° C. for 5 days. The progress of the binding is monitored by analyzing a small amount of the reaction mixture on Superros 6 (Pharmacia) gel filtration chromatography by high speed protein liquid chromatography (FPLC). The reaction ends when the height of the peak emanating from the column's pore volume (representing high molecular weight conjugate) is constant. The conjugate is purified by chromatography on Biogel A, 0.5M (Bio-Rad Laboratories, Richmond, Calif.) As described above (33). The protein content of the vaccine is determined by Lowry et al. (25) method using bovine serum albumin as a standard. Carbohydrate content is measured by Dubois et al. (11) using purified type II polysaccharide as standard.

Type V uncensory polysaccharides are purified from GBS type V strain 1169-NT1 cells by the above method for purified type III polysaccharides (33). The binding of the V polysaccharide to the monomer TT is carried out using the technique described above to bind the TT to the GBS III polysaccharide (33).

Natural V-type polysaccharides have a relative molecular weight of 200,000 daltons. Analysis of this material by H-nuclear magnetic resonance spectroscopy at 500 MHZ confirmed the native V-type structure (2) and the absence of group B antigen (26).

In order to introduce reactive aldehyde groups into the terminal sialic acid residues of the V polysaccharide to bind to the protein, the V polysaccharide is moderately oxidized with sodium meta-iodate (18). This process converts the sialic acid group moiety of the polysaccharide into a handic acid with an 8 carbon sialic acid homologue, 5-acetimido-3,5-dideoxy-D-galactosyloxytul (33). The proportion of oxidized sialic acid groups is determined by gas chromatography-mass spectroscopy of sialic acid groups and their oxidized derivatives.

The oxidized V-type polysaccharide is bound to monomer TT (Institute Armand Frappier, Montreal, Canada) by reductive amination as described above (33).

TT is purified into monomers by gel filtration chromatography as described above (33). In brief, 8.6 mg of periodate-treated V-polysaccharide and 8.7 mg of purified TT are dissolved in 0.6 ml of 0.1 M sodium carbonate, pH 8.2.

Sodium cyanoborohydride (31.5 mg) is added to the mixture and incubated at 37 ° C. for 10 days. The progress of binding is monitored by analyzing a small amount of the reaction mixture on Superros 6 (Pharmacia) gel filtration chromatography by high speed protein liquid chromatography (FPLC). The reaction is terminated when the height of the peak flowing out of the pore volume of the column (representing a high molecular weight conjugate) is constant. The binder is performed by chromatography on 2.6 X 91 cm Sephacryl S-300 HR (Pharmacia) using 0.01 M phosphate, 0.15 M NaCl, pH 7 and 0.01% thimerosal as effluent buffer. The protein content of the vaccine is determined by the method of Lowry et al. (25), based on bovine serum albumin. Carbohydrate content is measured by Dubois et al. (11) using purified type II polysaccharide as standard.

Vaccine II-TT Vaccine and V-TT Vaccine. Three groups of New Zealand white rabbit females (Millbrook Farms, Amherst, Mass.), Each consisting of about 3 kg of rabbits, are complete with 50 ug of unbound native type II polysaccharide or II-TT vaccine to a total volume of 2 ml each. Emulsify with Freund's adjuvant and inoculate subcutaneously by injecting it into 4 areas of rabbit's back. These animals are given a further stimulus injection (50 ug) of the vaccine made with incomplete Freund's adjuvant on days 20 and 41. Serum is taken from each animal on days 0, 20, 34, 41, 55 and 70, sterile filtered and stored at -80 ° C.

Approximately 3 kg of New Zealand White Rabbit females (Millbrook Farms, Amherst, Mass.) Were mixed subcutaneously in 4 parts of the rabbit's back by mixing 50 ug of GBS V-TT coupled vaccine with alum to a total volume of 2 ml. Inoculate. These animals are subjected to additional stimulation injection (50 ug) of vaccines made with incomplete alum at 22 and 42 days. Serum is taken from each animal on days 0, 35, 56 and 91, sterile filtered and stored at -80 ° C.

ELISA. GBS type II-specific rabbit antibodies are quantified by diluting 1 / 3,000 by goat anti-rabbit IgG (Tago Inc. Burlingham, Calif.) Bound to alkaline phosphatase and enzyme-linked immunosorbent assay (ELISA). The microtiter plate (Immulon 2; Dynatech Laboratories, Inc., Chantillty, Va.) Is coated with 100 ng of the refined type II polysaccharide bound to poly-L-lysine per well as described above (15, 33). If the antibody titer was diluted logarithmically and recorded, and reached as a result of the well is 0.5 A ₄₀₅ containing the reference serum at a dilution of 1/800 (all GBS 18RS21 cells), rabbit anti-serum generated by) A is ≥ 0.3 is ₄₀₅ do. The amount of antibody specific for the protein portion of the binding vaccine is determined by ELISA using plates coated with monomeric TT (100 ng per well). TT-specific IgG titers were recorded as logarithmic dilution, resulting in A _405> 0.3 after 35 minutes of addition of the substrate, p-nitrophenyl phosphate (Sigma 104 phosphatase substrate tablets; Sigma Chemical Co.).

GBS V-specific rabbit antibodies are quantified by diluting 1 / 3,000 by goat anti-rabbit IgG (Tago Inc. Burlingham, Calif.) Bound to alkaline phosphatase and ELISA. Microtiter plates (Immulon 2; Dynatech Laboratories, Inc., Chantillty, Va.) Are coated with 100 ng of the purified V-type polysaccharide bound to poly-L-lysine per well as described above (15). If the antibody titer was diluted logarithmically and recorded, and reaches a result 1/3000 diluted serum standard well of 0.3 A for ₄₀₅ containing (rabbit antiserum generated to the full V-type strain 1169-NT1) at the A ₄₀₅ is ≥ 0.3.

Isolation of IgG and IgM from Immune Rabbit Serum. Protein A-agarose affinity column chromatography (Pierce Chemical Co., Rockford, III.) Was immunoglobulin obtained on day 70 from 0.5 ml of packed immune rabbit serum derived from II-TT vaccine as described above. IgG and IgM) were used to separate (28). Isolation of antibodies was performed by goat anti-rabbit IgG (γ and short chain specific; Tago) diluted to 1/500 and goat anti-rabbit IgM (μ chain specific; Sera-Lab diluted to 1/200). , Westbury, NY), using a type II polysaccharide-coated ELISA plate.

Competitive ELISA. The specificity of rabbit serum induced with II-TT vaccine is determined by competitive ELISA with homologous (type II) and heterologous (types Ia and III) polysaccharides as inhibitors. Epitope specificity of vaccine-derived pooled rabbit serum (taken at 70 days) is tested with β-O-methylgalactopyranose and natural and desialylated type II polysaccharides as antibody binding inhibitors. Natural type II polysaccharides are desialized by treatment with 6% acetic acid at 80 ° C. for 2 hours. The polysaccharide inhibitor is serially diluted 4 fold and mixed with pooled equivalent (75 μl) rabbit serum taken 70 days after inoculation with the II-TT vaccine. This mixture (100 μl) is added to a type II polysaccharide-coated ELISA well. Alkaline phosphatase-binding anti-rabbit IgG is diluted 1 / 3,000 to use as second antibody. The results are given as follows:% inhibition = [- in the absence (A ₄₀₅ A ₄₀₅ when the absence of inhibitor inhibitor) / inhibitor A _405] X 100.

Serotype specificity of GBS V-TT rabbit antiserum is assessed using GBS type-specific polysaccharides Ia, Ib, II, III, V and VI. ELISA wells were coated at 100 ng / well concentration with GBS V-type polysaccharide bound to poly-L-lysine. Rabbit antiserum for V-TT vaccine was diluted to 1 / 6,000 and incubated with polysaccharide inhibitors, respectively. Incubated antiserum is added to the ELISA wells and the plates are treated as described above with goat anti-rabbit IgG alkaline phosphatase conjugate (1/3000 dilution), and substrate (28).

Antibody-mediated killing of GBS in vitro. The ability of vaccine-derived rabbit antibodies to opsonize GBS cells for sequential killing by human peripheral blood leukocytes is measured by an in vitro omsonin phagocyte assay (7, 8).

Reaction assays for measuring opsonophagocytic activity of GBS-type active antibodies include 1.5 × 10 ⁷ leukocytes (WBC) in 300 μl of buffer; Form V GBS strain CJB 111 of 3 × 10 ⁶ CFU in 50 μl; Human complement (uptake into GBS V strain cells) (50 μl); Heat-inactivated antibody-anti V-TT (50 μl); It is performed by incubation in modified Eagle's Medium (50 μl). The difference in GBS colony forming unit (CFU) is measured by incubation at 37 ° C. for 1 hour by standard plate counting.

Passive Protection of Mice with Vaccine-Induced Rabbit Antibodies. 0.2 ml of serum (70) pooled from rabbits vaccinated with type II polysaccharides or II-TT vaccine in groups of 10 of each Swiss-Webster non-facial mouse female (Taconic Farms, Gernamtown, NY), each of 18 to 20 g of mouse. Intraperitoneally The titer measured by ELISA of pooled serum obtained on day 70 from rabbits immunized with type II polysaccharide was 100, and 12,800 for II-TT vaccine. Unbound TT was induced in controls of five mice receiving pooled immunization rabbit serum or pooled antiserum (27). After 24 hours, mice are administered type II strain 18RS21 (1.5 × 10 ⁵ CFU per mouse) in a total dose of 1.0 ml Todd-Hewitt broth. Dosage doses for each strain are predetermined to kill> 90% mice of similar weight and age. Survival mice are counted daily for three consecutive days.

Statistical analysis. Fisher's extract test is used to compare different rabbit serum abilities to passively protect mice against lethal GBS infection.

[result]

Preparation and Composition of II-TT Vaccine and V-TT Vaccine. GBS II-TT vaccines and GBS V-TT vaccines are prepared by the method described above for the structure of the GBS III-binding vaccine (33). Controlled periodate oxidation of type II GBS polysaccharides modified 7% sialic acid groups of the polysaccharides as measured by gas chromatography-mass spectroscopy analysis. The monomer TT covalently bound to the modified sialic acid site of the type II polysaccharide by reductive amination. The purified II-TT vaccine contains 32% (wt / wt) protein and 68% (wt / wt) carbohydrate. The final yield of the II-TT vaccine was 7.8 mg, or 39%.

Controlled periodate oxidation of V-type GBS polysaccharides modified about 8%, 25% and 50% sialic acid groups of the polysaccharides. The monomer TT was covalently bound to the modified sialic acid site of the V-type polysaccharide by reductive amination. The polysaccharide is oxidized to modify about 8% of the sialic acid group to form a conjugate having a low molar ratio of polysaccharide to protein. Polysaccharides modified with 25 or 50% of the terminal sialic acid form a conjugate with a more preferred molar ratio of polysaccharide to protein. GBS type V polysaccharides modified with 25% sialic acid groups are used to prepare binding vaccines having the biochemical properties of Table 1 below.

Immunogenicity of II-TT Vaccine and V-TT Vaccine in Rabbits. The immunogenicity of the II-TT vaccine and native type II polysaccharides was compared in rabbits. An increase in type II-specific antibodies was seen after the first dose of II-TT vaccine (Table 2). The antibody response was increased by the administration of additional vaccine promoting doses. At day 41, antibody levels remained unchanged or slightly increased after the second promotion dose and were maintained for the remainder of the trial period (Table 2). In contrast to the II-TT vaccine, unbound native GBS type II polysaccharides failed to elicit specific antibody responses (Table 2). In animals vaccinated with II-TT, antibodies to TT also developed, resulting in an approximately 3-log ₁₀ increase relative to pre-immune levels.

^A value of ^a 100 indicates antibody titer ≤ 100. The figures are the average of two measurements. Rabbits were vaccinated by subcutaneous injection of 50 μg of the vaccine emulsified with complete Freund adjuvant on day 0.

^b A promoter was administered, including an incomplete Freund's adjuvant.

The immunogenicity of the V-TT vaccine (polysaccharide 25% oxidized) was also measured in rabbits. Antibody (lgG) levels increased after the first and second doses of V-TT vaccine and were maintained during the test (Table 3).

Antigen Characteristics of Vaccine-Induced Rabbit Serum. The binding of the polysaccharide with the protein carrier should not modify the important antigenic epitopes found in the natural forms of polysaccharides. The specificity of the II-TT vaccine-derived antibodies was tested by competitive ELISA using homologous and heterologous GBS polysaccharides as inhibitors. Natural type II polysaccharides at a concentration of 450 ng / ml inhibited 50% of rabbit antibody binding induced by II-TT vaccine. GBS type Ia and type III polysaccharides did not inhibit the binding of serum induced by II-TT vaccine even at concentrations of 500 μg / mg (FIG. 2). These results demonstrate serotype specificity for the target antigen of the II-TT vaccine-derived antibody and indicate preservation of the antigenic epitope of the polysaccharide portion of the binding vaccine.

To confirm that epitopes affected by sialic acid are maintained during the manufacture of the II-TT vaccine, natural and desialylated type II polysaccharides are used in competitive ELISA as binding inhibitors of rabbit antibodies derived from II-TT vaccines. It was. Desialylation of the polysaccharide was carried out with 6% acetic acid at 80 ° C. for 2 hours. Quantitative removal of sialic acid groups was confirmed using thiobarbituric acid assay (32) using N-acetylneuraminic acid (Sigma) as standard. K _av of natural Ⅱ type polysaccharide before acid treatment was 0.49, whereas, K _av of the acid-treated polysaccharide is the 0.52 on a Superose 6 FPLC column (LKB-Pharmacia, Sweden), which side chain sialic make up 20% of the native polysaccharide by weight The loss of acid groups indicates a slight decrease in the molecular size of the polysaccharides. 200 μg / ml of desialyl GBS type II polysaccharide inhibited 33% of antibody binding of the II-TT vaccine to native type II polysaccharides. Desialylation by the II-TT vaccine antiserum or relatively low recognition of nuclear type II polysaccharides was confirmed by immunoelectrophoresis gels, where a sedimentation band formed of native type II polysaccharides appeared, but not in type II nuclei. Not shown. The binding of the II-TT vaccine antiserum to natural type II polysaccharides cannot be inhibited with β-O-methylgalac topyranose used in a concentration range of 0.01 to 10 mg / ml (not shown).

Specificity of GBS type V-TT vaccine-induced antibodies is tested by competitive ELISA using homologous and heterologous GBS polysaccharides as inhibitors. Natural type V polysaccharides effectively inhibit the binding of rabbit antibodies induced with type V-TT vaccines (FIG. 5). GBS Type Ia, Type Ib, Type II, Type III and Type VI do not inhibit the binding of sera induced by Type V-TT vaccine (FIG. 5). These results show the specificity of the Type V-TT vaccine to induce antibodies to the target antigen.

In vitro activity of GBS vaccine-derived antibodies. The in vitro ability of the immune system to opsonize GBS to kill by human peripheral blood leukocytes correlates with the protective efficacy against GBS in animal protection experiments (27, 28). Antibodies generated in three rabbits vaccinated with II-TT increased the killing of GBS type II strain 18RS21 ≧ 1.8 log ₁₀ (Table 4). Rabbit serum prior to immunization or serum from rabbits vaccinated with native GBS II polysaccharides or unbound TT failed to increase GBS killing in vitro (Table 4). Vaccine-induced rabbit antibodies increase killing by human blood leukocytes of two GBS type II clinical isolates (strains S16 and S20) ≧ 1.8 log ₁₀ compared to pre-immunized rabbit serum (Table 5). Rabbit sera for type II-TT vaccines fail to increase the killing of heterologous (types la and III) GBS strains in vitro and are considered serotype specific (Table 5).

^The reaction mixture contains the serum to be tested (1% final assay concentration), type II GBS-absorbing human serum, human peripheral blood leukocytes, and type II GBS 18RS21 as complementary ingredients. The figures are the average of two measurements.

^b Rabbit blood cells were collected after the first dose of natural II polysaccharide in complete Freund's adjuvant.

^a CFU at log 60 (log ₁₀ ) -0 CFU at log ₁₀ (log ₁₀ ). The reaction mixture contains serum to be tested, type II GBS-absorbing human serum, human peripheral blood leukocytes, and type II GBS 18RS21 as complementary ingredients. The figures are the average of the two trials. ND not tested.

Protein A-affinity-purified IgG and IgM are obtained from pooled sera that induce type II-TT vaccines (28). The specificity of each Ig fraction is confirmed by ELISA as a class-specific secondary antibody. A ₄₀₅ S of the IgM and IgG fractions (diluted to 1/100) were 0.384 and 0.009 with μ-chain-specific conjugates, respectively and 0.086 and 2.367 with goat anti-rabbit IgG (γ and short chain specific), respectively. IgM and IgG isolated were tested for their ability to increase opsonin killing of type II GBS by human blood leukocytes. Unfractionated sera that induced type II-TT vaccine were diluted 1: 100, and killing of type II GBS by an equivalent 1: 100 dilution of the IgG fraction from the same serum increased 1.65 ± 0.22 and 0.95 ± 0.09 log _10, respectively. . In contrast, type II GBS is not killed but opsonophages in the presence of IgM-enriched fractions from serum which induce preimmune serum (-0.39 ± 0.13 log ₁₀ ) and type II-TT vaccine (-0.28 ± 0.09 log ₁₀ ) Increased in the sexual assay.

The ability of the GBS V-TT vaccine-induced rabbit antiserum to increase the killing of GBS-V type strain (CJB III) by human blood leukocytes was assessed by in vitro opsonine phagocyte assay (8). As shown in FIG. GBS V-TT antiserum at a concentration of 10% efficiently titrates GBS V strain CJB III compared to the control. The reaction conditions of the control group were as follows: complement without antiserum (10%); Antiserum without complement (10%); Antiserum (1%) and complement.

Mouse protection assay. To test the in vivo protective ability of vaccine-derived antibodies, mice are passively immunized with pooled II-TT vaccine serum (day 70) prior to administration of type II GBS 18RS21. Prior to that, the dosage is determined such that 90-100% of the test mice are lethal. Complete (100%) protection was achieved in the group of mice receiving sera that induced the GBS II-TT vaccine, whereas only one of five mice receiving sera prior to vaccination survived (Table 6). There were no survivors among mice receiving sera from rabbits vaccinated with unbound type II polysaccharide or unbound TT (Table 6).

^A mice received a 90% lethal dose (1.5 × 10 ⁵ CFU) DML GBS.

^b Pooled serum samples from 3 rabbits.

^c Lethality was measured 72 hours after administration.

^d P = 0.0037 was compared to preimmunization (day 0) values.

First, the binding method (18) used as the GBS type III polysaccharide developed in meningitis bacteria can be applied to all GBS capsular polysaccharide antigens because they all contain sialic acid. However, unlike other GBS polysaccharides having sialic acid as the terminal saccharide of the di- or trisaccharide side chain, GBS II polysaccharides have repeating units having sialic acid as a single sugar in one of the two monosaccharide side chains (9, 20). ).

In constructing a GBS type II binding vaccine, 7% of the sialic acid groups of the type II polysaccharides were oxidized and used to bind the polysaccharides to TT.

The purified II-TT vaccine was flowed into the pore volume of a Superros 6 column (molecular weight)> 10 ⁶ ) and consisted of 68% (wt / wt) carbohydrate and 32% (wt / wt) protein.

Adjuvant type II-TT vaccines have immunogenicity relative to unbound native type II polysaccharides in rabbits and fail to generate type II polysaccharide-specific antibodies. Two of the three immunized rabbits respond strongly three weeks after a single administration of the II-TT vaccine. Optimal type-specific antibodies were obtained in all three rabbits three weeks after the promoter of the II-TT vaccine. In addition, no increase in type II-specific antibody titer was seen after the third administration of the II-TT vaccine. In vitro and in vivo experimental results indicate that the antibody causing the II-TT vaccine has functional activity against type II GBS. Serum from each of the three rabbits vaccinated with II-TT increased the in vitro killing of type II GBS by human peripheral white blood cells and provided a non-myologic mouse that was fully (100%) protected against lethal doses of type II GBS. do. The II-TT vaccine antiserum showed opsonizing activity against homologous GBS strains (18RS21, S16, and S20) but not the heterologous GBS serotypes (Ia and III) tested.

While native type II polysaccharides inhibit the binding of the II-TT vaccine antiserum to type II polysaccharide-coated ELISA wells, desialylated type II polysaccharides exhibit a <40% inhibition rate even when used at a concentration of 200 μg / ml. Indicated. This result suggests that the important antigenic determinants of type II polysaccharides depend on the presence of sialic acid residues. This result confirms the results obtained by rabbit antiserum that induces the entire type II induction (21). However, ELISA inhibition experiments using β-O-methylgalactospyranose as inhibitors indicate that rabbit antiserum that induces II-TT vaccine does not contain galactose-specific antibodies. Inhibition of the binding of II-TT vaccine antiserum to type II natural polysaccharides cannot be achieved even with β-O-methylgalactosphyranos at a concentration of 10 mg / ml. Therefore, side chain galactose does not appear to be an immunodominant epitope of type II polysaccharide when the polysaccharide binds to TT. These results are in contrast to galactose side chains, which appear to be one of the two immunodominant sites of type II polysaccharides along sialic acid-dependent epitopes in immunochemical studies conducted with rabbit sera that induce the entire type II GBS organism (12, 21). to be. The entire type II GBS cells used in the studies (12, 21) and not cultured under pH-controlled conditions may, to some extent, have polysaccharides lacking sialic acid. Under these conditions, side chain galactose may be the major antigenic epitope. The raw material of the type II polysaccharide used to synthesize the II-TT vaccine is a type II GBS culture maintained at pH 7.0; The final analysis confirmed that sialic acid constitutes -20% (wt / wt) of polysaccharides. The possibility that binding type II polysaccharide to TT changes the form of the polysaccharide may render the galactose epitope recognized by the host immune system unusable. II-TT vaccine-derived rabbit antibodies are devoid of galactose-specific antibodies, but they function fully in vitro and in vivo for type II GBS organisms. Neither chemical modification of several sialic acid residues nor sequential binding of TT to these sites alters the important antigenic epitopes required to elicit functional type II-specific antibodies. IgG purified from rabbit serum causing II-TT vaccine promotes in vitro killing of type II GBS by human leukocytes, which not only increases the immunogenicity of type II polysaccharides by binding it to TT, but also functional IgG antibodies Suggest that is generated.

GBS type vaccinated polysaccharides have a repeating unit structure composed of trisaccharide backbones containing two different side chains in adjacent sugar groups. (Reference 35; FIG. 4). In one of the GBS type V-TT binding vaccine formulations, 25% of the sialic acid groups of the longer side chain (ie, trisaccharide group) of the type V polysaccharide were oxidized. The oxidized sialic acid group is used as the site for binding the V polysaccharide to TT. The purified Type V-TT vaccine consists of 38% (wt / wt) of TT and 62% (wt / wt) of carbohydrate. Type V-TT vaccine mixed with alum has been shown to be immunogenic in rabbits. A strong response was observed after the first and promoter administration of the Type V-TT vaccine. The second promoter further increases type V-polysaccharide IgG titer. Serum specificity of the Type V-TT induction assay was shown in the competitive ELISA. Earthenware antisera developed against the V-TT vaccine have functional activity, as demonstrated by the ability to kill Type V GBS in vitro in the presence of human leukocytes and complement.

Like the III-TT vaccine, the II-TT vaccine exhibits improved immunogenicity in rabbits compared to active polysaccharides and is treated with opsonin in rabbits despite differences in polysaccharide structure, location of sialic acid on TT-bound polysaccharides, and vaccine composition. Induce active IgG. V-TT vaccines have also been identified that are immunogenic in rabbits and are capable of inducing antibodies functionally in vivo. It is predicted that the GBS polysaccharide-protein conjugate of this design ultimately constitutes a component of the multivalent GBS vaccine that may allow protection against GBS serotypes that are very frequently associated with human disease.

In addition, vaccines may be produced from the binding molecules of the present disease by adding the binding molecules to a pharmaceutically acceptable carrier. Preferably, the degree of cross-linking of the antigenic molecules of the invention is controlled to an extent sufficient to provide a vaccine that can be filter sterilized through a 0.2 micron filter. Such vaccines are preferably soluble. Immunization can be effective by injecting the vaccine once in the recipient mammal, if necessary, followed by sequential injection of the promoter. Sufficient doses of the vaccine to be administered may be based on polysaccharide amounts: A combined vaccine of the polysaccharide component in the range of 3-80 μg may be administered.

[Reference Moonhyun]

Applicants believe that this final embodiment is combined with the previous embodiment to illustrate the overall feasibility and advantages of the method of the present invention.

However, these numerous features and advantages of the present invention have been described in detail in conjunction with the detailed description of the structure and function of the invention. This disclosure is by way of example only in terms of details, in particular in terms of shape, size and arrangement issues, order and time of steps. Changes can be made within the broad principles expressed to a sufficient extent in a broad general sense of the terms set forth in the appended claims of the present invention.

Adams, W. G., J. Kinney, A. Schuchat, C. Collier, C. Papasian, H. Kilbride, F. Riedo, and C. Broome, 1991, Program Abstr. 31st Intersci. Conf. Antimicrob. Agents Chemother., Abstr. 1056.

2.Avery, O.T., and W.F. Goebel, 1931. Chemoimmunological studies on conjugated carbohydrate-proteins. V. The immunological specificity of an antigen prepared by combining the capsular polysaccharide of type III pneumococcus with foreign protein. J. Exp. Med. 54: 437-447.

3. Baker, C.J., and M.S. Edwards, 1990, Group B streptococcal infections, p. 742-811, In J.S. Remington and J.O. Klein (ed.), Infectious diseases of the fetus and newborn infant. The W.B. Saunders Co., Philadelphia.

4. Baker, C.J., M.S. Edward, and D.L. Kasper, 1981, Role of antibody to native type III polysaccharide of group B Streptococcus in infant infection, Pediatrics 68: 544-549.

5. Baker, C. J., and D. L. Kasper, 1976. Correlation of material antibody deficiency with susceptibility to neonatal group B streptococcal infection, N. Engl. J. Med. 294: 753-756.

6. Baker, C. J., and D. L. Kasper, 1985. Group B streptococcal vaccines. Rev. Infect. Dis. 7: 458-467.

7. Baker, C.J., M.A. Rench, M.S. Edwards, R.J. Carpenter, B.M. Hays, and D. L. Kasper, 1988. Immunization of pregnant women with a polysaccharide vaccine of group B Streptococcus. N. Engl. J. Med. 319: 1180-1220.

8. Baltimore, R.S., D.L. Kasper, C.J. Baker, and D.K. Goroff, 1977. Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci. J. Immunol. 118: 673-678.

9. De Cueninck, B. J., T.F. Grebar, T.K. Eisenstein, R.M. Swenson, and G.D. Schockman, 1983. Isolation, chemical composition, and molecular size of extracellular type II and type Ia polysaccharides of group B. streptococci. Infect. Immun. 41: 527-534.

10. Dillon, H.C., S. Khare, and B.M. Gray, 1987. Group B streptococcal carriage and disease: a six-year retrospective study. J. Pediatr. 110: 31-36.

11. Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebars, and F. Smith. 1956. Colormetric method for the determination of sugars and related substances. Anal. Chem. 28: 350-356.

12. Freimer, E.H. 1967. Type-specific polysaccharide antigens of group B streptococci, J. Exp. Med. 125: 381-392.

13. Gelfand, H.M., J.P. Fox, D.R. LeBlanc, and L. Elveback, 1960. Studies on the development of natural immunity to poliomyletis in Louisiana.

V. Passive transfer of polioantibody from mother to fetus, and natural decline and disappearance of antibody in the infant. J. Immunol. 85: 46-55.

14. Goebel, W.F., and O.T. Avery, 1931. Chemo-Immunological studies on conjugated

carbohydrate-proteins. Ⅳ. The synthesis of the p-aminobenzyl ether of the soluble specific substance of type III pneumococcus and its coupling with protein. J. Exp. Med. 54: 431-436.

15. Gray, B.M. 1979. ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes. J. Immunol. Methods 28: 187-192.

16. Hobbs, J. R., and J.A. Davis, 1967. Serum gamma-G globulin levels and gestational age in premature babies. Lancet 493: 757-759.

17. Insel, R.A., and P.W. Anderson, 1986.

Oligosaccharide-protein conjugate vaccines induce and prime for oligoclonal IgG antibody responses th the Haemophilus Influenzae bcapsular polysaccharide in human infants. J.Exp. Med. 163: 262-269.

Jennings, H. J., and C. Lugowski, 1981.

Immunochemistry of groups A, B, and C meningococcal polysaccharide tetanus toxoid conjugates. J. Immunol. 127: 1011: 1018.

Jennings, H. J., C. Lugowski, and F.E. Ashton, 1984, Conjugation of meningococcal Iipopolysaccharide R-type oligosaccharides to tetanus toxoid as a route to a potential vaccine against group B Neisseria meningitidis. Infect. Immun. 43: 407-412.

20. Jennings, H.J., K.G. Rosell, E.

Katzenellenbogen, and D.L. Kasper. 1983.

Structural determination of the capsular polysaccharide antigen of type II group B Streptococcus. J. Biol. Chem. 258: 1793-1798.

21.Kasper, D.L., C.J. Baker, B. Galdes, E.

Katzenellenbogen, and H. J. Jennings. 1983.

Immunochemical analysis and immunogenicity of the type II group B streptococcal capsular polysaccharide. J. Clin. Invest. 72: 260-269

22. Lagergard, T., J. Shiloach, J.B. Robbins, and R.

Schneerson. 1990. Synthesis and immunocological properties of conjugates composed of group B Streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid. Infect. Immun. 58: 687-694.

23. Lancefield, R. C. 1972. Cellular antigens of group B streptococci, p. 57-65. In L.W.

Wannamaker and J.M. Matson (ed.), Streptococci and streptococcal diseases: recognition, understanding, and management. Academic Press, Inc. New York.

24. Lancefield, R. C., M. Mccarty, and W. N. Everly.

1975. Multiple mouse-protective antibodies directed against group B streptococci. J. Exp. Med. 142: 165-179.

25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951.Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275

26. Michon, F., E. Katzenellenbogen, D.L. Kasper, and H. L. Jennings. 1987. Structure of the complex group-specific polysaccharide of group B Streptococcus. Biochemistry 26: 476-486

27.Paoletti, L. c., D. L. Kasper, F. Michon, J.

Difabio, K. Holme, H.J. Jennings, and M.R. Wessels. 1990. An oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. J. Biol. Chem. 265: 18278-18283.

28.Paoletti, L.C., D.L. Kasper, F. Michon, H. J.

Jennings, T.D. Tosteson, and M.R. Wessels.

1992. Effects of chain length on the immunogenicity in rabbits of group B Streptococcus type III oligosaccharide-tetanus toxoid conjugates. J. Clin. Invest. 89: 203-209.

29. Schneerson, R. O. Barrera, A. Sutton, and J.B. Robbins. 1980. Preparation, characterization and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J. Exp. Med. 152: 361-376.

30. Svenson, S. B. and A. A. Lindberg. 1979.

Coupling of acid-labile Salmonella specific oligosaccharides to macromolecular carriers. J. Immunol. Methods 25: 323-335.

31. Walsh, J. A., and S. Hutchins. 1989. Group B streptococcal disease: its importance in the developing world and prospect for prevention with vaccines. Pediatr. Infect. Dis. J. 8: 271-276.

32. Warren, L. 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234: 1971-1975.

33. Wessels, M.R., L.C. paoletti, D.L. Kasper, J.L. Difabio, F. Michon, K. Holme, and H. J. Jennings. Immunogenicity in animals of a polysaccharide-protein conjugate vaccine against type III group B Streptococcus, J. Clin. Invest. 86: 1428-1433.

34. Zigterman, J.W.J., J.E.G. van Dam, H. Snippe, F. T. M Rottsveel, M. Jansze, J. M. N. Willers, J.P. Kamerling, and J.F.G. Vliegenthart. 1985.

Immunogenic properties of octasaccharide-protein conjugates derived from Klebsiella serotype II capsular polysaccharide. Infect. Immun. 47: 421-428.

35. Wessels, M.R., J.L.D; Fabio, V. J. Benedi, D.L. Kasper, F. Michon, J-R. Brisson, J. Jelinkova and H. J. Jennings 1991. Structural Determination and Immunochemical Characterization of the Type V Group B Streptococcus Capsular Polysaccharide. Journal of Biological Chemistry 266: 8714-8719.

While many embodiments of the invention have been described above, they may be modified to provide other embodiments that utilize the methods of the invention. Therefore, it is intended that the scope of the invention be defined by the claims appended hereto, rather than by the specific embodiments presented in the Examples.

Claims (40)

Two or more side chains comprising an acetylmeric polysaccharide and a protein component derived from group B streptococcus type II or V bacteria, each of which has a sialic acid group attached to the terminal of the polysaccharide component, respectively, through a secondary amine linkage. And the molecular weight of the polysaccharide is at least 5000 Daltons. The binding molecule of claim 1, wherein 5 to 50% of the terminal sialic acid group of the polysaccharide is capable of binding to the protein before the binding. The binding molecule of claim 2, wherein 5 to 25% of the terminal sialic acid group of the polysaccharide is capable of binding to the protein before the binding. 4. The binding molecule according to claim 3, wherein 5-10% of the terminal sialic acid group of the polysaccharide is capable of binding to the protein before the binding. 5. The binding molecule of claim 4, wherein the polysaccharide is Group B Streptococcus type II and wherein 7% of the terminal sialic acid group of the polysaccharide is capable of binding to the protein. The binding molecule of claim 1, wherein the antigenic binding molecule can be filter sterilized through a 0.2 micron filter. The binding molecule of claim 1, wherein the polysaccharide component has a molecular weight of about 5,000 to 1,000,000 Daltons. The binding molecule of claim 1, wherein the polysaccharide component has a molecular weight of about 50,000 to 500,000 Daltons. The binding molecule of claim 1, wherein the polysaccharide is Group B Streptococcus V-type and about 25% of the terminal sialic acid groups of the polysaccharide are bound to the protein. 9. The binding molecule of claim 8, wherein the polysaccharide component has a molecular weight of about 100,000 to 300,000 Daltons. 10. The binding molecule of claim 9, wherein the polysaccharide component has a molecular weight of about 200,000 Daltons. 2. The binding molecule of claim 1, wherein the protein is selected from the group consisting of tetanus toxin, diphtheria toxin and CRM (Cross Reactant) 197. (a) periodate oxidizing a Group B streptococcus type II or V anaerobic polysaccharide to a degree sufficient to introduce an aldehyde group to two or more terminal sialic acid groups bound to the polysaccharide backbone; (b) binding the oxidized group B streptococcus type II or V anaerobic polysaccharide to the protein via reductive amination to produce a secondary amine bond between the anaerobic polysaccharide and the protein. A method for producing a binding molecule of Group B streptococcus type II or V anticapillary polysaccharide and protein. The method of claim 13, wherein 5 to 50% of the sialic acid group in the polysaccharide is oxidized to form an aldehyde group bondable to the protein. 15. The method of claim 14, wherein 5-25% of the sialic acid group in the polysaccharide is oxidized to form an aldehyde group bondable to the protein. 16. The method of claim 15, wherein 5-10% of the sialic acid group in the polysaccharide is oxidized to form an aldehyde group bondable to the protein. 17. The method of claim 16, wherein said polysaccharide is Group B Streptococcus type II and wherein 7% of the sialic acid groups in the polysaccharide are oxidized to form an aldehyde group capable of binding to a protein. The method of claim 13, wherein 5 to 50% of the sialic acid groups in the polysaccharide are covalently bound to the protein. 19. The method of claim 18, wherein 5-25% of the sialic acid group in the polysaccharide is covalently bound to the protein. 20. The method of claim 19, wherein 5-10% of the sialic acid group in the polysaccharide is covalently bound to the protein. 20. The method of claim 19, wherein said polysaccharide is Group B Streptococcus Type II and wherein about 5% of the sialic acid group of the polysaccharide is covalently bound to the protein. The method of claim 13, wherein the polysaccharide is Group B Streptococcus V-type and wherein about 25% of the sialic acid group of the polysaccharide is covalently bound to the protein. A binding molecule produced by the method according to claim 13. A vaccine comprising the binding molecule according to claim 1. A multivalent vaccine comprising a binding molecule according to claim 1 and at least one other immunogenic molecule capable of causing the production of antibodies against pathogenic agents other than group B Streptococcus type II. 26. The method of claim 25, wherein the other immunogenic molecule is in a pathogenic agent selected from the group consisting of Group B Streptococcus Types Ia, Ib, II, III, IV and V, Haemophilus influenza b and E. coli K1. Multivalent vaccine, characterized in that it can cause the production of antibodies to. 27. The method of claim 26, wherein the other immunogenic molecule is an antibody against a pathogenic agent selected from the group consisting of Group B Streptococcus Types Ia, Ib, II, III, IV, Haemophilus influenza b and E. coli K1. Multivalent vaccine characterized in that it can cause production. 28. The method of claim 27, wherein the other immunogenic molecule is capable of causing the production of an antibody against a pathogenic agent selected from the group consisting of Group B Streptococcus Types Ia, Ib, II, III and E. coli K1. To the vaccine. 29. The multivalent vaccine of claim 28, wherein the other immunogenic molecule is capable of inducing the production of antibodies against pathogenic agents selected from the group consisting of Group B Streptococcus Types Ia, Ib, II, and III. . Mammalian subjects other than humans, comprising administering an immunogenic binding molecule to a female to produce an antibody that can pass through to the pup in an amount sufficient to prevent infection of the pup at birth. Wherein the binding molecule is composed of an antimolecular polysaccharide and a protein component selected from the group consisting of type II and V group B streptococcus bacteria, and two or more side chains at which the sialic acid group of the polysaccharide component is bound to the terminal A method for immunization against babies before birth, each linked via a primary amine bond, and wherein the polysaccharide has a molecular weight of at least 5,000 Daltons. 31. The method of claim 30, wherein the vaccine is administered before the female is pregnant and a booster is administered to immunize the female, if necessary. 33. The method of claim 30, wherein the vaccine is administered during pregnancy. An immunization method comprising administering a vaccine comprising a binding molecule according to claim 1 to a mammal other than a human body in an immunogen amount. An immunization method comprising administering a vaccine comprising the binding molecule of claim 1 to a mammal, except for a human body, which may be infected by group B streptococcus type II or V. 35. The method of claim 34, wherein said immune system is administered to a subject having a compromised immune system. 36. The method of claim 35, wherein the lowering of the immune system is due to a disease selected from the group consisting of cancer or diabetes. 37. The method of claim 36, wherein the vaccine is administered with a pharmaceutically acceptable carrier. A method for immunization against bovine mastitis characterized in that the vaccine comprising the binding molecule according to claim 1 is administered to the dairy cow in an immunogenic amount. 31. The method of claim 30, wherein said polysaccharide is derived from Group B Streptococcus type II bacteria. 32. The method of claim 30, wherein said polysaccharide is derived from Group B Streptococcus V bacteria.